Application suite image analysis software

Intestinal samples from the proximal jejunum and distal ileum were fixed with 10% formalin. After tissue processing, all segments were embedded in paraffin wax. The tissues were sectioned with a 5 μm thickness and stained with hematoxylin and eosin stain.
The specimens were examined and photographed under a light microscope with a DC490 digital camera (Leica, Wetzlar, Germany) by two histologists who were blinded to the study. The villus height and crypt depth for each specimen were measured in 10 villi and 10 crypts in the jejunum and ileum sections using the Leica Application Suite image analysis software (Leica, Wetzlar, Germany). Five random fields were examined under 20x objective by two investigators and injury in the intestinal mucosal tissues in all groups was graded semiquantitatively according to Chiu's classification [21 (link)]: 0, normal mucosal villi; 1, subepithelial space at the tips of the villi; 2, moderate elevation of the epithelial layer from the lamina propria; 3, massive epithelial elevation extending down the sides of the villi (a few tips may be denuded); 4, denuded villi with the lamina propria exposed and dilated capillaries; and 5, disintegration of the lamina propria, haemorrhage, and ulceration.

To determine intestinal histology, three paraformaldehyde-fixed intestinal segements (from duodenum, jejunum, and ileum) were dehydrated and embedded in paraffin. Five-μm sections were cut and then stained with hematoxylin and eosin. Intestinal histology was determined using a light microscope (Leica, Germany) with Leica Application suite image analysis software (Leica, Germany). Only vertically oriented villi and crypts were measured [12 (link)]. Histological indices, such as villus height (from the tip of the villi to the villus crypt junction), villus width at half-height, and crypt depth (defined as the depth of the invagination between adjacent villi) were determined from ten adjacent villi [33 (link)]. The villus:crypt ratio and villous surface area were calculated. All intestinal histological analysis was done by the same person, who was blinded to the treatments.